This invention relates to a cDNA which encodes tumor-associated antigen and to the use of the cDNA and the encoded protein in the diagnosis and treatment of inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer.
Phylogenetic relationships among organisms have been demonstrated many times, and studies from a diversity of prokaryotic and eukaryotic organisms suggest a more or less gradual evolution of molecules, biochemical and physiological mechanisms, and metabolic pathways. Despite different evolutionary pressures, the proteins of nematode, fly, rat, and man have common chemical and structural features and generally perform the same cellular function. Comparisons of the nucleic acid and protein sequences from organisms where structure and/or function are known accelerate the investigation of human sequences and allow the development of model systems for testing diagnostic and therapeutic agents for human conditions, diseases, and disorders.
Cancers, or malignant tumors, which are characterized by continuous cell proliferation and cell death, can be classified into three categories: carcinomas, sarcomas, and leukemia. Recent reports show that approximately one in eight women contracts breast cancer and that the risk of prostate cancer is about 9.5% among men over 50 years of age (Helzlsouer (1994) Curr Opin Oncol 6: 541-548; Harris et al. (1992) N Engl J Med 327:319-328). Cancer cells have been shown to exhibit unique gene expression, and many cancer-specific genetic markers, tumor antigens, have been identified.
Tumor antigens are surface molecules that are differentially expressed in tumor cells relative to non-tumor tissues. Tumor antigens make tumor cells immunologically distinct from normal cells and provide diagnostic and therapeutic targets for human cancers. Several monoclonal antibodies have been identified which react specifically with cancerous cells such as T-cell acute lymphoblastic leukemia and neuroblastoma (Minegishi et al. (1989) Leukemia Res 13:43-51; Takagi et al. (1995) Int J Cancer 61: 706-715). In addition, the discovery of high level expression of the HER2 gene in breast tumors has led to the development of therapeutic treatments (Liu et al. (1992) Oncogene 7: 1027-1032; Kern (1993) Am J Respir Cell Mol Biol 9:448-454).
Tumor antigens have been characterized either as membrane proteins or as altered carbohydrate molecules of glycoproteins or glycolipids on the cell surface. A multigene family encoding type III integral membrane proteins which traverse the cell membrane four times has been identified (Wright and Tomlinson (1994) Immunol Today 15:588-94). The transmembrane 4 superfamily (TM4SF) proteins are found predominantly in cells of hematopoietic origin and in tumors and include a number of platelet and endothelial cell membrane proteins; CD9 (lung adenocarcinoma antigen MRP-1), the platelet and melanoma-associated antigen CD63, leukocyte surface glycoproteins, CD53, CD37, CD63, and R2, the tumor associated antigen TAPA-1 (CD81), the colonal carcinoma antigen CO-029, mink lung epithelial protein TI-1, and the tumor-associated antigens L6 and SAS, a gene amplified in human sarcomas (Wright and Tomlinson, supra; Jankowski et al. (1994) Oncogene 9:1205-1211). These proteins all share 25-30% amino acid sequence identity.
In the TM4SF proteins, the N- and C-termini are intracellular and the major hydrophilic domain, located between transmembrane domains 3 and 4, is extracellular. TM4SF proteins are most conserved in their transmembrane and cytoplasmic domains and most divergent in their hydrophilic extracellular domains which contain N-linked glycosylation sites. The high level of conservation in the transmembrane and cytoplasmic domains suggests an effector/signaling function. The divergence of the extracellular domains suggests that these hydrophilic domains provide functions specific to each protein such as ligand binding or protein-protein interaction (Wright and Tomlinson, supra).
A number of TM4SF proteins have been implicated in signal transduction, control of cell adhesion, and regulation of cell growth and proliferation (Wright and Tomlinson, supra; Jankowski, supra). Expression of some TM4SF proteins is associated with a variety of tumors and is altered when cells are activated or dividing. Other TM4SF proteins are implicated in cell growth due to their association with tumor cells. For example, CD9, CD53, and CD82 are upregulated when lymphocytes are activated while the expression of CD37 is abolished when B cells are activated. Although CD9 is not expressed on resting B and T lymphocytes, it is a marker for 90% of non-T acute lymphoblastic leukemia cells and for 50% of acute myeloid and chronic lymphoid leukemias. Anti-CD9 antibodies inhibit the motility of a variety of cancer cell lines and inhibit the metastatic potential of the mouse BL6 cell line (Miyake and Hakomori (1991) Biochem 30:3328-3334). Similarly, CD63 is not expressed on normal tissue melanocytes, but it is expressed in early stage melanoma. Another member of the TM4SF superfamily, the L6 surface antigen, is differentially expressed on lung, breast, colon, and ovarian carcinomas. This antigen is an attractive target for therapeutic intervention due to its high level of expression on malignant cells (Marken et al. (1992) Proc Natl Acad Sci USA 89: 3503-3507; Marken et al. (1994) J Biol Chem 269: 7397-7401).
The discovery of a cDNA encoding tumor-associated antigen similar to the tumor-associated L6 antigen satisfies a need in the art by providing compositions which are useful in the diagnosis and treatment of inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer.
The invention is based on the discovery of a cDNA encoding tumor-associated antigen (TUAN) which is useful in the diagnosis and treatment of inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer.
The invention provides an isolated cDNA comprising a nucleic acid sequence encoding a protein having the amino acid sequence of SEQ ID NO:1. The invention also provides an isolated cDNA or the complement thereof selected from the group consisting of a nucleic acid sequence of SEQ ID NO:2, a fragment of SEQ ID NO:2 selected from SEQ ID NOs:34, and a variant of SEQ ID NO:2 selected from SEQ ID NOs:5-7. The invention additionally provides a composition, a substrate, and a probe comprising the cDNA, or the complement of the cDNA, encoding TUAN. The invention further provides a vector containing the cDNA, a host cell containing the vector and a method for using the cDNA to make TUAN. The invention still further provides a transgenic cell line or organism comprising the vector containing the cDNA encoding TUAN. The invention additionally provides a fragment, a variant, or the complement of the cDNA selected from the group consisting of SEQ ID Nos:2-7. In one aspect, the invention provides a substrate containing at least one of these fragments or variants or the complements thereof. In a second aspect, the invention provides a probe comprising a cDNA or the complement thereof which can be used in methods of detection, screening, and purification. In a further aspect, the probe is a single-stranded complementary RNA or DNA molecule.
The invention provides a method for using a cDNA to detect the differential expression of a nucleic acid in a sample comprising hybridizing a probe to the nucleic acids, thereby forming hybridization complexes and comparing hybridization complex formation with a standard, wherein the comparison indicates the differential expression of the cDNA in the sample. In one aspect, the method of detection further comprises amplifying the nucleic acids of the sample prior to hybridization. In another aspect, the method showing differential expression of the cDNA is used to diagnose inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer. In another aspect, the cDNA or a fragment or a variant or the complements thereof may comprise an element on an array.
The invention additionally provides a method for using a cDNA or a fragment or a variant or the complements thereof to screen a library or plurality of molecules or compounds to identify at least one ligand which specifically binds the cDNA, the method comprising combining the cDNA with the molecules or compounds under conditions allowing specific binding, and detecting specific binding to the cDNA, thereby identifying a ligand which specifically binds the cDNA. In one aspect, the molecules or compounds are selected from aptamers, DNA molecules, RNA molecules, peptide nucleic acids, artificial chromosome constructions, peptides, transcription factors, repressors, and regulatory molecules.
The invention provides a purified protein or a portion thereof selected from the group consisting of an amino acid sequence of SEQ ID NO:1, a variant having at least 85% identity to the amino acid sequence of SEQ ID NO:1, an antigenic epitope of SEQ ID NO:1, and a biologically active portion of SEQ ID NO:1. The invention also provides a composition comprising the purified protein and a pharmaceutical carrier. The invention further provides a method of using the TUAN to treat a subject with inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer comprising administering to a patient in need of such treatment the composition containing the purified protein. The invention still further provides a method for using a protein to screen a library or a plurality of molecules or compounds to identify at least one ligand, the method comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules or compounds are selected from DNA molecules, RNA molecules, peptide nucleic acids, peptides, proteins, mimetics, agonists, antagonists, antibodies, immunoglobulins, inhibitors, and drugs. In another aspect, the ligand is used to treat a subject with inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer.
The invention provides a method of using a protein to screen a subject sample for antibodies which specifically bind the protein comprising isolating antibodies from the subject sample, contacting the isolated antibodies with the protein under conditions that allow specific binding, dissociating the antibody from the bound-protein, and comparing the quantity of antibody with known standards, wherein the presence or quantity of antibody is diagnostic of inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer.
The invention also provides a method of using a protein to prepare and purify antibodies comprising immunizing a animal with the protein under conditions to elicit an antibody response, isolating animal antibodies, attaching the protein to a substrate, contacting the substrate with isolated antibodies under conditions to allow specific binding to the protein, dissociating the antibodies from the protein, thereby obtaining purified antibodies.
The invention provides a purified antibody which binds specifically to a protein which is expressed in inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer. The invention also provides a method of using an antibody to diagnose inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer comprising combining the antibody comparing the quantity of bound antibody to known standards, thereby establishing the presence of inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer. The invention further provides a method of using an antibody to treat inflammatory and cell proliferative disorders, in particular, Crohn""s disease and colon cancer comprising administering to a patient in need of such treatment a composition comprising the purified antibody and a pharmaceutical carrier.
The invention provides a method for inserting a heterologous marker gene into the genomic DNA of a mammal to disrupt the expression of the endogenous polynucleotide. The invention also provides a method for using a cDNA to produce a mammalian model system, the method comprising constructing a vector containing the cDNA selected from SEQ ID NOs:2-7, transforming the vector into an embryonic stem cell, selecting a transformed embryonic stem cell, microinjecting the transformed embryonic stem cell into a mammalian blastocyst, thereby forming a chimeric blastocyst, transferring the chimeric blastocyst into a pseudopregnant dam, wherein the dam gives birth to a chimeric offspring containing the cDNA in its germ line, and breeding the chimeric mammal to produce a homozygous, mammalian model system.